CN1649607A - A process for the extraction of anthocyanins from black rice and composition thereof - Google Patents

Abstract

A process of extracting a composition comprising anthocyanins from black rice (Oryza sativa L) comprises separating an outer layer from a starchy endosperm in de-hulled black rice; adding a solution of at least one organic solvent and an acid to the separated outer layer; filtering and removing the solvent and the acid from the separated outer layer to produce a pigment fraction; separating constituents of the pigment fraction; and collecting the anthocyanin composition therefrom. This composition is useful in enhancing and/or preserving the stability of HDL-C and the atherogenic lipioproteins such as LDL-C, VLDL-C, and IDL-C from oxidation, in preventing, reducing, eliminating or ameliorating injuries due to oxidative stress, and in preventing, reducing, eliminating or ameliorating the development of atherosclerotic lesions and inflammation associated therewith.

Description

A kind of method and compositions thereof of from Black rice, extracting anthocyanin

Invention field

The present invention relates to treat or prevent animal, particularly people's cardiovascular disease and underlying diseases thereof, comprise the method for symptom such as atherosclerosis, oxidative stress and unusual lipidemia or disease.

Background of invention

The latest developments of science and technology help to improve human life quality and life-saving, atherosclerosis is a reason of cardiovascular disease (" CVD "), its prevention is not also fully solved, and is still the U.S., Europe and the primary cause of death of Asia Desk region-by-region (1).Atherosclerosis is a kind of by inherited genetic factors and environmental factors (as diet and life style) the caused degenerative process that influences each other.Up to now research prompting, cholesterol may work in atherosclerosis, and it forms atheromatous plaque in blood vessel, block cardiac muscle at last or to the blood supply of brain or extremity, this depends on the position (2,3) of speckle in arterial tree.Summary points out that individual total serum cholesterol reduces 1% can make the danger that coronary artery events takes place reduce 2% (4).Statistically, average serum cholesterol reduction by 10% (for example being reduced to 5.3mmol/L from 6.0mmol/L) can prevent 100,000 examples dead (5) every year in the U.S..Therefore, relevant with the rising of low density lipoprotein, LDL (LDL) cholesterol concentration with T-CHOL hyperlipemia is an important risk.

Research shows that also the plasma concentration of high density lipoprotein (HDL) cholesterol is low to be an important risk factor (6) of progression of atherosclerosis, and high level has protectiveness.

Lipoprotein is the complex that lipid and protein combine by non-covalent bond.Every kind of lipoprotein type has distinctive quality, chemical composition, density and physiological action.The shell irrelevant with density or granular size, that circulation lipid comprises cholesteryl ester and triglyceride core and is made of phospholipid, free cholesterol and apolipoprotein.The assembling of apolipoprotein and lipoprotein and secrete relevant, provide structure integrity, activate the lipoprotein modification enzyme, and be the part of multiclass receptor and memebrane protein.The lipoprotein classification of finding in blood plasma comprises HDL, LDL, intermediate density lipoprotein (IDL) (IDL) and very low density lipoprotein (VLDL) (VLDL).

Every type lipoprotein all has distinctive apolipoprotein and forms or ratio.Topmost apolipoprotein is apolipoprotein-A I (apoA-I) among the HDL, accounts for the about 70% of protein quality, and apoA-II accounts for other 20%.The ratio of apoA-I and apoA-II can determine function and the atherosclerosis character of HDL.Circulation HDLGranule comprises the multiphase mixture of disc and spheroidal particle, and quality is the 200-400 kilodalton, and diameter is 7-10nm.

HDL is one of main lipoprotein classification that works in the plasma lipid transhipment, multiple function is arranged in vivo, comprise the antiport cholesterol, cholesterol molecule substrate, transhipment element (clusterin), transhipment paraoxonase, prevention lipoprotein oxidation and the adrenal cells selectivity picked-up to cholesterol of trooping is provided for bile acid is synthetic.Comprise cholesterol, cholesteryl ester, triglyceride, phospholipid and fatty acid with the bonded main lipid of DHL.

How antiatherogenic in order to understand HDL better is, brief explanation Tremulous pulse Atherosis processBe necessary.Atherosclerosis begins in the time of in LDL is captured to blood vessel wall.The oxidation of this LDL causes Mononuclear cellCombine with the endothelial layer of blood vessel wall.These mononuclear cells are activated, and move to the endothelium gap, and they are converted into macrophage there, make the further oxidation of LDL.The LDL of oxidation is by the scavenger on the macrophage ReceptorBe ingested, cause Foam cellFormation.Propagation and migration by arterial smooth muscle cell produce a fibrous cap, thereby produce atheromatous plaque.

It is necessary to the liver transhipment from extrahepatic tissue that HDL is a cholesterol, and the bile acid secretion that it forms as free cholesterol or by cholesterol in liver is in bile.This process need several steps.The first step is to form newborn or preceding β HDL granule in liver and intestinal.Excessive cholesterol passes cell membrane by the effect of ABCA I transport protein and moves in the newborn HDL.Lecithin-cholesterol acyltransferase. (LCAT) is converted into cholesteryl ester with cholesterol, and the HDL with new life is converted into sophisticated HDL subsequently.The cholesterol of esterification is transferred to the apo-B that contains lipoprotein by cholesterol ester transfer protein (CETP) from HDL then, and the latter is by a large amount of receptor picked-ups in the liver.Newborn HDL is by liver tg lipase and phospholipid transfer protein regeneration, and the continuation circulation.Except the cholesterol from peripheral cells, HDL also accepts the cholesterol from LDL and erythrocyte membrane.The another one mechanism of cholesterol antiport may relate to cholesterol in less film of cholesterol and the passive diffusion between HDL or other acceptor molecule.

HDL by the cholesterol antiport effect and may prevent atherosclerosis by stoping the LDL oxidation.There are several HDL desmoenzymes to participate in this process.Paraoxonase (PON1), LCAT and platelet activating factor PAF-AH (PAFAH) all participate in by the phospholipid hydroperoxide that produces in the hydrolysis LDL oxidizing process, and effect successively, stop the accumulation of oxidation lipid among the LDL.These enzymes are responsible for antioxidation and the anti-inflammatory character of HDL.

Although hypercholesterolemia is CVD patient's a important risk factor, also must consider other risk factor, increase (7) as oxidative stress and serum homocysteine.The modification of these harmful components will help forming treatment and develop or have development CVD danger but do not suffer from the patient's of hypercholesterolemia new method (8) in the tremulous pulse.

Verified, oxidative stress has important function (9) in the startup of the multiple chronic disease that comprises atherosclerosis and development.Active oxygen composition (ROS) is general and natural existence in all aerobic species, the deutero-exogenous source that comes from that endogenous metabolism generates and environment is originated.ROS is differentiable hardly reactive molecule, and they damage biomacromolecule easily, comprises DNA, protein, carbohydrate and lipid (10).It is relevant with the development of the atherosclerosis of different phase and CVD that excessive or uncontrolled ROS generates, comprise vascular endothelial cell damage, foam cell formation, vascular smooth muscle hypertrophy, gene expression, vasculomotor reactive damage and speckle unstability (11,12a).

Under the situation that does not have enough endogenous antioxidant protections, the increase of free radical can cause cooxidation and the secondary lipid autoxidation product and the macromolecular reaction of nucleophilic of nucleophilic cellular component.The example of oxidative stress indicator comprises 8-hydroxyl deoxyguanosine, malonaldehyde, 4-hydroxyl aldehyde C-9 and lipid peroxide in target tissue and the blood, not only comprises the fatty acid oxidation product, and comprises cholesterol oxidation product.By remove from the cell antioxidant system of enzyme-specific and non-enzyme and detoxifcation ROS realizes control to oxidative stress comprehensively.For example, relevant with the detoxifcation of lipid and oxygen-derived free radicals enzyme antioxidant comprises Cu/Zn superoxide dismutase (SOD; Cytoplasm), catalase (peroxisome) selenium-glutathion peroxidase/reductase redox reaction cyclophorase (GSH-Px in Cytoplasm and the mitochondrion and GSHG-Red) and non-selenium glutathione-S-transferase (Cytoplasm).Non-enzyme cell antioxidant comprises alpha-tocopherol, ascorbic acid and beta-carotene.

Evidence external and the interior research of body is pointed out, and the oxidative modification of LDL is relevant with atherosclerotic generation, and increases the weight of its clinical manifestation.The concrete product of cholesterol oxidation is identified (12b) in the atheromatous plaque.Therefore, can influence the antioxidant that the non-enzyme antioxidant activity of enzyme antioxidant activity or complement improves, those of those diet source particularly, thus the possible medicine that ROS prevents to develop into atherosclerosis and CVD produced as preventing, be subjected to suitable concern.

Just the someone advises with rice Diet Therapy CVD decades ago.Before 50 years, reported that edible white rice can bring high blood pressure down, and reduced human hypercholesterolemia (13).Yet, many different types of rice are arranged.Up to the present, human edible modal rice is white rice (more than 85%), secondly is red rice and Black rice.The back both mainly in South Asia, Greece, Italy and U.S.'s plantation.Europe is than the edible more coloured rice (14) in South Asia, and coloured rice has had very long edible history in China, is considered to a kind of health food of physical strength reinforcing.Therefore, be called " blood strengthens rice " or " medicine rice " usually.

The pigment level of Black rice is every 100g rice 1mg, and every 100g contains 3mg vitamin C and 0.2mg riboflavin, compares with the rice that does not contain pigment and contains more ferrum, calcium and phosphorus.At the Ka Lala of India, kind Navara is considered to have medicinal properties, is used for recovering paralytic's nerve; Oridine is a kind of alkaloid that exists in the rice, has certain anti-neurosis characteristic when impure.

Research before the Black rice field concentrates on the Black rice pigment as coloring agent (China Patent No. 93109627.8), the preparation and the purposes of adding the drink and food (China Patent No. 93109627.8) of Black rice pigment, and full Black rice and red rice diet are to the effect (15) of serum lipid and aorta speckle.

One object of the present invention is elimination or reduces chemical compound known in the art and the shortcoming and defect of compositions aspect treatment and prevention CVD and all underlying diseases (comprising atherosclerosis, oxidative stress and dyslipidemia disease) thereof.Another object of the present invention is to handle Black rice, to optimize its various curative effects.

Summary of the invention

One aspect of the present invention provides a kind of method for compositions that contains anthocyanin of extracting from Black rice (Oryza sativa L), comprising:

A) skin of separation, hulling Black rice and starchy endosperm;

B) in separated outer layer, add at least a organic solvent and sour solution;

C) from separated outer layer, filter and remove and desolvate and acid, chromogenesis fraction (fraction);

D) component of separation pigment fraction; With

E) therefrom collect the anthocyanin compositions.

The present invention also comprises the anthocyanidin-3-O-glucoside that contains from Black rice and the compositions of peonidin-3-O-glucoside.

The present invention also provides a kind of treatment or the cardiovascular disease of prevention animal and the method for underlying diseases (comprising atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress) thereof, comprises the compositions of using by extracting method preparation of the present invention.

The present invention also provides the method for a kind of treatment or prevention animal cardiovascular disease and underlying diseases (comprising atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress) thereof, comprises using the anthocyanidin-3-O-glucoside that contains from Black rice and the compositions of peonidin-3-O-glucoside.

A key of the inventive method is that the therapeutic activity composition anthocyanin of finding Black rice can extract from isolating rice granule skin on most convenient ground.The distribution of anthocyanin is obviously relevant with the particulate degree of depth, and outermost layer contains the anthocyanin of top level.The previously known method of extracting anthocyanin from Black rice is not recognized the advantage that this is important.

These effects and some other remarkable advantage are described hereinafter in more detail.

The accompanying drawing summary

The present invention describes by following non-limitative drawings, wherein:

Fig. 1 is the figure that shows composition on the Oryza sativa L. granule cross section;

Fig. 2 is the Bio-Gel P-2 elution chromatography figure of Black rice extract, and peak 1 and peak 2 are accredited as peonidin-3-glucoside and anthocyanidin-3-glucoside by LC/MS respectively subsequently;

Fig. 3 is that Bio-Gel Xian P-2 of Black rice extract takes off chromatogram;

Fig. 4 is the Bio-Gel P-2 elution chromatography figure of Black rice extract, and a peak is accredited as peonidin-3-glucoside by LC/MS subsequently;

Fig. 5 is the Bio-Gel P-2 elution chromatography figure of Black rice extract, and a peak is accredited as anthocyanidin-3-glucoside by LC/MS subsequently;

Fig. 6 shows that the Black rice extract is to the inhibiting figure of DPPH free radical;

The curve chart of Fig. 7 shows the influence that 37 ℃ of following Black rice extracts form conjugated diene in the liposome peroxidation reaction of hydrogen peroxide free yl induction;

Fig. 8 shows the agarose gel electrophoresis result of Black rice extract effect aspect the negative charge that suppresses oxidized modified LDL, the 1st road=natural LDL wherein, the 2nd road=LDL+ bivalent cupric ion, 3-7 road=LDL+ bivalent cupric ion and Black rice extract, the 8th road=LDL+ bivalent cupric ion and EDTA;

The bar diagram of Fig. 9 shows that the external LDL oxidative modification of people LDL suppresses (CD and TBARS);

Figure 10 shows the agarose gel electrophoresis result of Black rice extract effect aspect the super coiled DNA fracture of prevention hydrogen peroxide free yl induction, S=super coiled DNA wherein, the 1st road=DNA+PBS, the 2nd road=DNA+AAPH, 3rd, 4,5 and 6 road difference=DNA+AAPH+1,10,25,100 μ g/ml Black rice extracts, the 7th, 8 road difference=DNA+AAPH+1,10 μ g/ml Trolox ^TM

Figure 11 shows that peonidin-3-glucoside and anthocyanidin-3-glucoside (10 μ g/ml) are combined in the agarose gel electrophoresis result of the dna break damage aspect effect that reduces the hydrogen peroxide free yl induction, wherein the 1st, 2 roads respectively=DNA of natural and oxidation, 3rd, 4,5,6 and 7 road difference=DNA+AAPH and 9/1,4/1,1/1,1/4 and 1/9 anthocyanidin-3-glucoside and peonidin-3-glucoside, the 8th road=DNA+AAPH and 10 μ g/mlTrolox ^TM

Figure 12 shows that the Black rice extract is preventing that hydrogen peroxide free radical (non-locus specificity) inducing DNA from producing the agarose gel electrophoresis result of the effect aspect the otch, the 1st road=DNA+PBS wherein, the 2nd road=DNA+ hydroxyl radical free radical initiator, 3rd, 4 road difference=DNA+ hydroxyl radical free radical initiator+1.7,17mg/ml Black rice extract (ground floor), 5th, 6 road difference=DNA+ hydroxyl radical free radical initiator+1.7, the particulate extract of the whole Black rice of 170mg/ml, the 7th, 8 road difference=DNA+ hydroxyl radical free radical initiator+0.17,1.7mg/ml Trolox ^TM

Figure 13 shows that the Black rice extract is preventing that hydroxyl radical free radical (locus specificity) inducing DNA from producing the agarose gel electrophoresis result of effect aspect the otch, the 1st road=DNA+PBS wherein, the 2nd road=DNA+ hydroxyl radical free radical initiator, 3rd, 4 road difference=DNA+ hydroxyl radical free radical initiator+1.7,17mg/ml Black rice extract (ground floor), 5th, 6 road difference=DNA+ hydroxyl radical free radical initiator+1.7, the particulate extract of the whole Black rice of 170mg/ml, the 7th, 8 road difference=DNA+ hydroxyl radical free radical initiator+0.17,1.7mg/ml Trolox ^TM

The bar diagram of Figure 14 shows the cell survival experiment of carrying out with the Black rice extract;

The bar diagram of Figure 15 shows that the Black rice extract is to the active inhibition of hepatic lipase;

Figure 16 shows the time dependent body weight of different disposal group mice;

Figure 17 shows not atheromatous plaque area on the same group.16 all laggard action pulse atherosclerosis degree quantitatively.By measure the area of atherosclerotic lesion in the hole with the oil red O stain lesion portion.Numerical value is meansigma methods ± SD, n=15.There is not the post of common letter to have significant difference, P＜0.01;

Figure 18 shows the influence of Black rice fraction of the present invention to the nitric oxide inhibition of bacteria lipopolysaccharide stimulation among the mouse macrophage RAW264.7; With

Figure 19 shows the agarose gel electrophoresis result of Black rice extract of the present invention to the influence of inducible nitric oxide synthase expression.

The preferred embodiments of the invention

Below describing in detail is intended to help those skilled in the art to implement the present invention.Yet this detailed description should not be regarded as limiting the scope of the invention inadequately.Under the condition that does not deviate from the spirit or scope of the present invention, those skilled in the art can modify and change embodiment described herein.

Extracting method

The invention provides a kind of method for compositions that contains anthocyanin of from Black rice (Oryza sativa L), extracting, comprise skin and the starchy endosperm of separation, hulling Black rice; In separated outer layer, add the solution of at least a organic solvent and acid; From separated outer layer, filter and remove and desolvate and acid, the chromogenesis fraction; The component of separating pigment fraction; Therefrom collect the anthocyanin compositions.

The raw material that is used for the inventive method is the Black rice of shelling.The shelling of Oryza glutinosa can be carried out with several different methods well known in the art.Be generally manual (smashing to pieces by hand) or mechanical dejacketing.The mechanical dejacketing machine has 3 kinds of main type: Engelberg mills, stone hulling machine and rubber hulling machines.The stone hulling machine is still common in Tropical Asia.The rubber hulling machine is common in Japan, stores the rice rather than the Oryza glutinosa of shelling in Japan, thereby saves the space.

The first step of the inventive method is a committed step, comprises skin and the starchy endosperm of separation, hulling Black rice.For better understanding, Fig. 1 has shown the basis on the Oryza sativa L. granule cross section.Remove the decapsidate composition in the shelling process, for example behind cellulose and the hemicellulose, what keep usually is peel, endotesta and the aleurone that is positioned at outside the starchy endosperm.Within the scope of the present invention, wish to separate and be somebody's turn to do outer " aleurone " layer, and therefrom extract the anthocyanin compositions.Up to now, whole Oryza sativa L. granule uses in the anthocyanin extracting method, but should never use by skin.

Outer and starchy endosperm separate preferably by physical separation, most preferably by the realization of milling.Already used polytype is milled in the production of coloured rice and subtractive process, also can use at this.In these current known productions, discard skin usually, because consumer buys the rice granule that shells, mills, polishes then.Within the scope of the present invention, reclaim the material of milling, and therefrom extract the anthocyanin compositions at last.

Many rice mills commonly used are arranged, and Engelberg is ground to multi-channel system from single channel.Within the scope of the present invention, can use to comprise manual manual technology of smashing to pieces, and utilize the machine that grinds and rub to mill.Most preferably, use a kind of mill that utilizes physics to scrape to rub.Satake USAInc. and Buhler AG provide the example of suitable mill.

Elongated grain needs the lower pressure of milling than thick (thick) grain, because their aleurone is thinner.Mill, all be removed up to all skins (comprising aleurone) basically.This layer comprises the particulate 5-15% of Oryza sativa L. usually.In a preferred form, as an index determining that mill processes stops, people can measure about Oryza sativa L. particulate 10%.In addition, people also can estimate the change of grain color, stop this mill processes when granule becomes light colored with respect to raw material.Can particle depth adjustment as required process.

The innovation that can utilize the industry of Japanese rice to introduce within the scope of the present invention comprises according to required and milling and degree that plumule rice is milled that microcomputer control is milled.A kind of plumule rice mill of introducing in 1976 uses gently to grind spreading under utmost point low-pressure, makes the plumule of 80% above grain be kept perfectly.Like this, can obtain higher business efficiency, because treat upward useful anthocyanin compositions with outer the extraction, particulate remainder can be used as commodity selling.

Table 1 shows the roughly analysis of the outer fraction of Black rice that complete Black rice and Shimzu mill are milled, and is used for comparison:

	Black rice (skin)	Black rice (complete granule)
			Protein g/100mg	????17	??10.0
Fat g/100mg	????9.8	??2.0
			Moisture g/100mg	????8.3	??11.0
Ash g/100mg	????7.6	??1.4
			Carbohydrate g/100mg	????57.0	??76.0
Energy Kilojoule/100mg	????1610	??1510

Obviously, the complete granule of every 100mg outer level proportion by subtraction contains the nutrient of higher concentration.

The comparative distribution of anthocyanin in the complete granule of table 2 demonstration Black rice and each fraction thereof

??? Fraction	????Mg/kg
		Complete granule	????1645±25
Ground floor	????13,567±802
		The second layer	????3850±876
Kernel	????70±9

Find that now the distribution of anthocyanin (combination of anthocyanidin-3-glucoside and peonidin-3-glucoside) is relevant with particle depth, isolating ground floor contains the most high-load anthocyanin from the starchy endosperm.This discovery provides optimizes the unique opportunity that anthocyanin reclaims.

Second step of the inventive method comprises the solution that adds at least a organic solvent and acid in separated outer layer.This organic solvent is preferably selected from alcohol, ketone, hydrocarbon and water.

Ketone is selected from has general formula R COR ¹Ketone, wherein R and R ¹It is alkyl.Alkyl is C preferably ₁-C ₆Group.Ketone most preferably is 2-acetone.Hydrocarbon can be selected from all C ₅-C ₁₀Hydrocarbon.Most preferably hydrocarbon is a hexane.Alcohol be selected from have general formula R-CHOHR, R-CH ₂The alcohol of OH and RCOH, wherein R is C ₁-C ₄Alkyl.Most preferably alcohol is methanol or ethanol.

Acid can be any nontoxic, food acids of being enough to reach required pH scope.Acid is preferably selected from hydrochloric acid, acetic acid, citric acid, tartaric acid and low-concentration sulfuric acid.The preferred acid that adds capacity, making pH is 1-4.Preferably separated outer layer was immersed in the solvent 1-10 hour, temperature is 25-40 ℃.Preferred solvent is 30/1 to 100/1 (volume/weight) with the ratio of rice.

The 3rd step of the inventive method comprises filters from separated outer layer and removes and desolvate and acid, the chromogenesis fraction.Although some other method obviously also within the scope of the present invention, for example vacuum drying and lyophilization preferably removes by filtrate extraction and desolvates, and removes disacidify by evaporation (for example rotary evaporation).

The 4th step of the inventive method comprises the component of separating and collecting pigment fraction with the 5th step.Preferably realize separating by classification.Most preferably, can use method (for example polyamide gels filtration) as column chromatography.In this stage, collect two main fraction after the classification, one contains anthocyanidin-3-glucoside, and another contains peonidin-3-glucoside.

Can determine fraction with high pressure liquid chromatography (HPLC), liquid chromatography/mass spectrometry method (LC/MS) as required after the separation.Fig. 2 is the Bio-Gel P-2 elution chromatography figure of Black rice extract, and the 1st peak and the 2nd peak are accredited as peonidin-3-glucoside and anthocyanidin-3-glucoside respectively with LC/MS subsequently.

Preferred Black rice kind that can be used according to the invention includes but not limited to: Wu Gong, Shang-nong, Hei Xiang Geng Nuo, Zhen Xi Hei Mi, Dong Bei Nuo149, Ao Yu Nuo 349, Hong280, Hong 463 and Xiang Xue Nuo.

The anthocyanin compositions

The present invention includes and contain the anthocyanidin-3-O-glucoside that derives from Black rice and the compositions of peonidin-3-O-glucoside.These compositionss comprise the compositions that extraction described herein and purification process produce, and wherein two kinds of compositionss (promptly not necessarily deriving from this method) that components in proportions is as mentioned below of de novo synthesis.

The structure of anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside is as follows:

Anthocyanidin-3-glucoside peonidin-3-glucoside

In a preferred embodiment, the present composition contains anthocyanidin-3-glucoside and peonidin-3-glucoside, and its ratio is 10: 1, and more preferably 6: 1, more preferably 3.5: 1 to 6.5: 1.Most preferably, this ratio is 4: 1.

Within the scope of the present invention, anthocyanidin-3-the glucoside and the peonidin-3-glucoside that are used for preparing these compositionss can extract from multiple source or obtain, include but not limited to Black rice, black fruit source (as blackberry and Ribes nigrum L.), Semen sojae atricolor, black seed such as Semen Sesami and black cereal such as rye (Secale cereale L.).In addition, other source comprises black currant and red rice.

In one embodiment of the invention, in order to obtain to have the compositions of aforesaid preferred anthocyanin ratio, a kind of most preferred method is the anthocyanin of " mixing " separate sources.Like this, people can be used to the high-caliber especially a kind of composition from a source, and the another kind of composition that needs can be rich in another source.

With antioxidant and/or plant sterol combination

In the another one embodiment, compositions of the present invention can mix, use jointly or with certain hour separate administration at interval using with one or more antioxidants.Suitable antioxidant includes but not limited to: vitamin E, beta-carotene, superoxide dismutase, catalase, glutathion peroxidase, glutathion reductase, tea catechin, chelating agen such as citric acid, EDTA, phenylalanine, phosphoric acid, tartaric acid and tryptophan; The chemical compound of preferential oxidation is as ascorbic acid, sodium sulfite and sodium sulfite; Water solublity chain terminating agent such as mercaptan and fat-soluble chain terminating agent such as alkyl gallates, ascorbic palmitate, tertiary butylated hydroquinone, butylated hydroxyanisol, Yoshinox BHT, hydroquinone, nordihydroguaiaretic acid and alpha-tocopherol.Can think that the combination startup of these antioxidants and anthocyanin compositions of the present invention also keeps useful antioxidation.

In the another one embodiment, the present composition can mix, use jointly or with certain hour separate administration at interval using with one or more sterols or stanol.

Term used herein " sterol " comprises all sterols without limitation, for example: sitosterol, campesterol, stigmasterol, brassicasterol (comprising Neospongosterol,dihydro-), desmosterol, chalinosterol, poriferasterol, chionasterol, ergosterol, coprostenol, codisterol, isofucosterol, 24-ethylidenecholest-5-en-3.beta.-ol., loyal paulownia sterol, nervisterol, lathosterol, asteriasterol, chondrillasterol, chondrillasterol, peposterol, avenasterol (avenasterol), different avenasterol (isoavenasterol), fecosterol, pollinastasterol, cholesterol, and all natural or synthesized form and derivants, comprise isomer.Term " stanol " is meant saturated or hydrogenant sterol, comprises their all natural or synthesized form and derivants, and isomer.Can think that the scope of the invention also comprises the modification to sterol and stanol, for example make it to comprise side chain.For example, obviously comprise 24 β-ethyl Dihydrocholesterol (24 β-ethylchlostanol), 24-α-ethyl-22-dehydrogenation Dihydrocholesterol (24-α-ethyl-22-dehydrocholstanol) in the scope of the invention.Can think that also when this description was had a question, unless otherwise indicated, term " sterol " comprised sterol and stanol.In a most preferred form, sterol is its saturated form, and is sitostamol.

These sterols and stanol that the present invention uses can be from multiple natural origins.For example, can from vegetable oil (comprising water plant) processing, obtain, as Semen Maydis oil and other vegetable oil, wheat germ oil, soybean extract, rice extract, rice chaff, rapeseed oil, Oleum helianthi, Oleum sesami and fish (with other marine products source) oil.They also can derive from for example ergosterol of fungus, or animal cholesterol for example.Therefore, the invention is not restricted to any sterol source.United States Patent (USP) series number 4,420,427 has been described and has been utilized solvent (as methanol) to prepare sterol by the plant slurry oil.In addition, of United States Patent (USP) series number 5,770,749, plant sterol and phytostanol also can be available from the by-products of tall oil resin or soap class, forestry operation, and this United States Patent (USP) is incorporated herein by reference.

Can think that the combination of these sterols and/or stanol and anthocyanin compositions of the present invention produces adduction at least, also may be collaborative and supplement therapy effect, particularly in lipid is regulated.Though be not subjected to any theoretical restriction the about mechanism of action, this may be because the lipid target difference of every kind of composition.For example, prove that sterol and stanol are the active drugs that reduces serum LDL-C, and they almost there be not effect aspect the raising Serum HDL-C.On the contrary, find that anthocyanin of the present invention and sterol compositions effectively and quite fully make HDL-C and LDL-C stablize and exempt from oxidation.In addition, anthocyanin composition and sterol composition are to the minimizing of atherosclerotic pathological development with stop to have supplementary function substantially.

Curative effect

In first embodiment of the present invention, a kind of treatment or the cardiovascular disease of prevention animal (preferred people) and the method for underlying diseases (comprising atherosclerosis and relevant with it inflammation and oxidative stress) thereof are provided, have comprised the compositions that contains anthocyanidin-3-glucoside and peonidin-3-glucoside from Black rice to animal administering therapeutic effective dose.

In another embodiment of the invention, a kind of treatment or the cardiovascular disease of prevention animal (preferred people) and the method for underlying diseases (comprising atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress) thereof are provided, comprise the compositions that contains anthocyanidin-3-glucoside and peonidin-3-glucoside that derives from Black rice to animal administering therapeutic effective dose, and optional one or more plant sterols or phytostanol and/or one or more antioxidants.

These compositionss comprise such compositions, wherein:

1) the anthocyanin composition derives from extraction described herein and purification process;

2) the anthocyanin composition is a de novo synthesis, and two kinds of anthocyanin components in proportions as described here; With

3) the anthocyanin composition extracts from more than one source and purification, for example Black rice, Semen sojae atricolor, black race's, blackberry, and the ratio of mixing the anthocyanidin-3-glucoside and the peonidin-3-glucoside that reach required.

Term " treatment is effectively " is used for limiting the amount of composition of using in order to reach one or more following purposes:

A) raising and/or maintenance HDL are for the stability of oxidation;

B) raising and/or maintenance LDL, VLDL or IDL are for the stability of oxidation;

C) raising and/or maintenance triglyceride (TG) are for the stability of oxidation;

D) prevent, reduce, eliminate or improve unusual fat disease;

E) prevent, reduce, eliminate or improve hypercholesterolemia, hypoalphalipoproteinemia;

F) prevent, reduce, eliminate or improve the development of atherosclerotic lesion;

G) development of inflammation that prevention, minimizing, elimination or improvement are relevant with the coronary artery disease development with cardiovascular disease;

H) prevention, reduce, eliminate or improve based on plasma D HL shortage or LDL, VLDL, Lp (a), β-VLDL, IDL or all the other lipoproteins are excessive or any symptom, disease or disorder that it is increased the weight of; With

I) prevent, reduce, eliminate or improve the damage that oxidative stress causes.

Can clearly be seen that by the following example, prove that compositions display powerful antioxidant of the present invention effect can effectively reduce the formation of atheromatous plaque by a large amount of detections.

Now find, when therapeutic administration, anthocyanin compositions of the present invention can:

1) enhancing and/or maintenance HDL-C and atherogenicity lipoprotein such as LDL-C, VLDL-C and IDL-C are for the stability of oxidation;

2) prevent, reduce, eliminate or improve the damage that oxidative stress causes;

3) prevent, reduce, eliminate or improve the development of atherosclerotic lesion; With

4) prevention, minimizing, elimination or improvement and atherosclerosis, coronary artery disease (CAD) and the relevant inflammation of cardiovascular disease (CVD).This is very crucial, because recent findings, because inflammation, CAD and CVD take place in obviously low danger crowd.Nearest evidence is also pointed out, and the inflammation of tremulous pulse may be an important indication of following heart attack and apoplexy.When health response damage or infection, be inflamed.

Now find, when treatment is used, anthocyanin of the present invention and plant sterol/phytostanol compositions can:

1) prevents, reduces, eliminates or improve the development of atherosclerotic lesion;

2) regulate or control plasma lipoprotein;

3) prevent, reduce, eliminate or improve multiple symptom and disease, include but not limited to: cardiovascular disease and underlying diseases thereof comprise the development of atherosclerosis, dyslipidemia symptom or disease, hypercholesterolemia, hypoalphalipoproteinemia, atherosclerotic lesion and toxic shock syndrome; With

4) preventing, reduce, eliminate or improving plasma D HL shortage or LDL, VLDL or IDL excessive is multiple symptom or disease basic or that it is increased the weight of.

Using method

Compositions of the present invention can be co-administered by any conventional method and medicine, nutriment, food, beverage etc.

Do not limit above-described universality, compositions of the present invention can be mixed with various carriers or adjuvant, directly uses or mix said composition in food, beverage, nutriment or medicine helping.In order to be familiar with the various possible carrier of carrying these compositionss, provide following Example.The dosage of compositions is with mode of movement, weight in patients and the state of an illness, the result who desires to reach and food additive and the known other factors of pharmaceutical formulation those skilled in the art.

Required effect described herein can obtain by multiple distinct methods.These compositionss can be co-administered by any operable conventional method and medicine, nutriment, food, beverage etc.

The amount that obtains the compositions that required effect needs depends on many factors certainly, as concrete composition distribution, method of application and the patient's of compositions the state of an illness.

1) pharmaceutical formulation:

Compositions of the present invention can directly be applied to the patient, perhaps uses with the form of pharmaceutical composition with suitable carrier or mixed with excipients.

The scope of the invention comprises uses drug acceptable carrier the present composition to be mixed with the dosage form that is suitable for systemic administration.By suitable selection carrier and suitable production method, the present composition particularly is formulated as the compositions of solution, can parenteral administration, and as intravenous injection.The enough drug acceptable carriers well known in the art of these compositionss energy easily are mixed with and are suitable for oral dosage form.These carriers can make the present composition be formulated as tablet, pill, capsule, liquid, gel, syrup, serosity, suspension etc., so that the patient who is treated is oral.

The pharmaceutical preparation that contains one or more present compositions comprises and contains the effective dose formulations of active ingredients that accomplishes the end in view.Those skilled in the art particularly according to detailed disclosure provided herein, can determine effective amount.

Except active component, these pharmaceutical compositions also can contain suitable drug acceptable carrier, include to be beneficial to excipient and the auxiliary agent that active component is processed into pharmaceutical formulation.For oral, the preparation of preparation can be the form of tablet, lozenge, capsule or solution.

Pharmaceutical composition of the present invention can be used known method production, for example conventional mixing, dissolving, granulating, lozenge formation, porphyrize, emulsifying, seals, holds back or vacuum lyophilization.

The pharmaceutical preparation that is used for parenteral administration comprises the aqueous solution of the reactive compound of water-soluble form.In addition, the suspension of reactive compound also can be made into suitable oily injection suspension.Suitable lipophilic solvent or carrier comprise fatty oil, as Oleum sesami, or synthetic fatty acid ester, as ethyl oleate or triglyceride, or liposome.The water injection suspension can contain the material that improves suspension viscosity, as sodium carboxymethyl cellulose, Sorbitol or glucosan.Randomly, suspension also can contain suitable stabilizing agent or improve the reagent of compound dissolution, with the highly spissated solution of preparation.

Being used for oral pharmaceutical preparation can followingly obtain: reactive compound is mixed with solid excipient, randomly grind the gained mixture, the processing granular mixture adds proper assistant in case of necessity, obtains tablet or lozenge nuclear afterwards.Particularly, appropriate excipients has: filler as sugar, comprises lactose, sucrose, mannitol or Sorbitol; Cellulosics is as corn starch, wheaten starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).Can add disintegrating agent in case of necessity, as crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt, as sodium alginate.

Lozenge nuclear has suitable coating.For this reason, can use spissated sugar juice, its optional arabic gum, Pulvis Talci, polyvinylpyrrolidone, carbopol gel, Polyethylene Glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture of containing.In order to identify or characterize the various combination of active compound doses, can in tablet or lozenge coating, add dyestuff or pigment.

Can oral pharmaceutical preparation comprise sucking fit (push-fit) capsule of making by gelatin, and the sealing soft capsule of making by gelatin and plasticizer (as glycerol or Sorbitol).The sucking fit capsule can contain and the blended active component of following ingredients: filler such as lactose, binding agent such as starch, and/or lubricant such as Talcum or magnesium stearate and stabilizing agent randomly.In soft capsule, reactive compound can dissolve or be suspended in the suitable liquid, as fatty oil, liquid paraffin or liquid macrogol.Also can add stabilizing agent in addition.

Oral liquid can be following form: example emulsion, syrup or elixir, perhaps can be used as dry products, and water or other appropriate carriers are rebuild before use.These liquid preparations can contain conventional additives, as suspending agent, and for example Sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel, hydrogenant edible fat; Emulsifying agent, for example lecithin, dehydrated sorbitol mono-fatty acid ester or arabic gum; Non-aqueous carrier (can comprise edible oil), for example almond oil, fractionated Oleum Cocois, grease are as glycerol, propylene glycol or alcoholic acid ester; Antiseptic, for example methyl parahydroxybenzoate or propyl ester or sorbic acid; When needing, flavoring agent commonly used or coloring agent.

Can expect within the scope of the invention, compositions of the present invention can be mixed in various conventional medicine preparations and the dosage form with usual excipients and/or diluent and stabilizing agent, as is used for oral, the tablet of buccal or tongue clothes (common with coating), capsule (snap fit capsule and soft capsule, be with or without other coating), powder, granule (comprising effervescent granule), pilule, microparticle, solution is (as micelle, syrup, elixir and drop), lozenge, pastille, the ampoule injection, Emulsion, microemulsion, ointment, unguentum, suppository, gel, the release dosage form of transdermal patch and improvement.

Be fit to be added to the present composition in the above-mentioned appropriate dosage forms, can animal (comprising the people) be used by oral, injection (intravenous, subcutaneous, intraperitoneal, Intradermal or intramuscular), part or alternate manner.

The concrete method of application of the present composition all depends on the purpose of application program in all cases.For existing symptom and disease, depend on severity of disease, and individual as mentioned above age, body weight and sex.Those skilled in the art can determine suitable dosage and use progress.

2) food/beverage/nutriment

In another kind of form of the present invention, the present composition can be impregnated in food, beverage and the nutriment, and the preventive use that is used to continue includes but not limited to following:

1) milk product-as cheese, milk and other milk drink, tablespread and milk mixture, ice cream and Yoghurt;

2) based on the product of corn-comprise corn (for example bread, edible face, cookies and cereal bar), no matter these article boil, toast or with other method processing;

3) sweet food-as chocolate, caked sugar, chewing gum, dessert, non-milk system (the Cool Whip for example that gravys with meat or vegetables poured over rice or noodles (toppings) ^TM), sherbet, sugar-coat and other filler;

4) beverage-ethanol or non-alcoholic beverage, comprising can happy other soft drink, fruit drink, food additives and for the food and drink material, is Boost as trade mark ^TMAnd Ensure ^TMBeverage;

5) other products-comprise the food of processing, as soup, ready-formed pasta sauces, prefabricated food etc.

Compositions of the present invention can not need further modification by directly mixing in food, nutriment or the beverage as technology such as mixing, infusion, injection, fusion, dispersion, emulsifying, submergence, injection and kneadings.In addition, these compositionss also can directly be added on the food or in the beverage by consumer before edible.This is simple, economic mode of administration.

Embodiment

The present invention describes by following non-limiting examples:

Embodiment 1: the extraction of anthocyanin with separate

From Wu Gong Black rice, isolate skin by the Shimzu Mill among the POS (Saskatchewan).The Black rice fraction is middle soaked overnight in containing the methanol of 1%HCl.Use Whatman filter paper filtering extract No. 4.Under 40 ℃, remove methanol by rotary evaporation.It is that the Bio-Gel P-2 post of water filling of pH2.5 is (on 2.5 * 45cm) that the pigment fraction that obtains is added to the acetic acid acidify.

This post is collected fraction (5ml/ pipe or 5min/ pipe) with being acidified to the flow velocity eluting of the water of pH2.5 with 1ml/min with fraction collector.Under 520nm, monitor sample.Collect two main fraction, under vacuum, concentrate in order to identifying.

Embodiment 2: the quantitative and evaluation of anthocyanin

Further use standard reference (anthocyanidin-3-glucoside and peonidin-3-glucoside) to analyze two main fraction of embodiment 1 by HPLC and LC/MS.HPLC is with being equipped with Waters Alliance 2690 systems of automatic sampler and post heater to carry out.The condition of HPLC is as follows: use Waters Xterra MS C18 post (2.1 * 50mm, 2.5 μ m) down at 40 ℃, inject 5 μ l, solvent orange 2 A is 100% methanol, and solvent B is 5% aqueous formic acid.The concentration of A dropped to 4% from 20% in 5 minutes.Flow velocity is set to 0.4ml/min.Use Waters996 PDA detector, wavelength set is 200-600nm.

Utilize with the link coupled electrospray mass spectrometry of Waters 2690 separation modules the Black rice fraction is carried out the LC/MS analysis.PDA shows, utilizes this Bio-Gel P-2 chromatography, obtains two sections (Fig. 3,4,5) that retention time and spectrum can both be distinguished.Fig. 3 shows fraction I and the fraction II after Bio-Gel P-2 separates.

Embodiment 3: the test of antioxidant activity DPPH free radical scavenging

With stable free radical 1, estimate by 1-biphenyl-2-picryl (" DPPH ") in alcoholic solution for the free radical scavenging activity of the ground floor of Black rice, the second layer and whole particle.When with other free radical scavenger, during particularly basic antioxidant (hydrogen donor) reaction, DPPH will change into the non-free radical form.Utilize spectrometry to measure the disappearance of DPPH in the alcoholic solution.

Fig. 6 shows every layer of influence that the DPPH free radical is suppressed with whole particle.Obviously, under each concentration that detects, ground floor is given prominence to.

Embodiment 4: antioxidant activity liposome model

Estimate the free radical scavenging activity of anthocyanin compositions of the present invention in the liposome model, wherein the oxidation of phospholipid is by 2, and the thermal decomposition effect of 2-azo two (∝-amidine propane) dihydrochloride (" AAPH ") is induced.By the formation of 37 ℃ of following conjugated diene hydroperoxides of continuous monitoring, carry out this test.

Fig. 7 shows that the anthocyanin compositions of variable concentrations prevents the usefulness of conjugated diene hydroperoxide formation, is the most effective more than the 10 μ g/ml.

Embodiment 5:LDL inhibition of oxidation

Anthocyanin compositions of the present invention is prevented that the oxidation of LDL from assessing.Because the downward modulation of ox-LDL is modified, the oxidative modification of LDL is a committed step in the progression of atherosclerosis.The particulate modification of LDL causes macrophage to form foam cell, forms load (laden) and speckle subsequently on blood vessel endothelium.Therefore, minimizing LDL is considered to an important step in the cardiovascular disease prevention.Remove transition metal ions by dialysis, with Cu ²⁺Ion is 37 ℃ of oxidations of incubation a period of time startup LDL altogether down.With distinct methods monitoring LDL oxidation, comprise the formation of agarose gel electrophoresis, conjugated diene and thiobarbituric acid reaction substrate.Result among Fig. 8 shows, adds the distance that anthocyanin compositions of the present invention has shortened electrophoretic migration, shows that the negative charge increase of oxidized modified LDL is suppressed.

And the formation of conjugated diene and thiobarbituric acid reaction substrate also reduces (Fig. 9), and prompting generation first and secondary fat peroxidating product also is subjected to the inhibition of anthocyanin compositions of the present invention.Also assessed the oxidation of HDL in addition.Measure as agarose gel electrophoresis, add the HDL modification that anthocyanin compositions of the present invention has stoped the hydrogen peroxide free yl induction.

Embodiment 7:DNA inhibition of oxidation

Studied the inhibitory action of anthocyanin compositions to the inductive DNA oxidation of oxidative free radical (as hydrogen peroxide free radical and hydroxy radical) (site-specific and non-site-specific).Figure 10 shows that the hydrogen peroxide free radical causes the super coiled DNA chain to disappear, and adds anthocyanin compositions or standard anti-oxidant Trolox this damaged portion is recovered.Unite interpolation anthocyanidin-3-glucoside and peonidin-3-glucoside obtains similar effects (Figure 11) with different ratios.

Black rice extract of the present invention or compositions suppress non-locus specificity and the inductive dna break of locus specificity hydroxy radical (Figure 12 and 13).In the hydroxy radical model, by the Fenton reaction generation hydroxy radical of ascorbic acid mediation.Discovery Black rice extract can be removed the hydroxyl radical free radical that produces in the locus specificity model, also proves its chelating transition metal ions in locus specificity hydroxyl radical free radical model.

Embodiment 8: the cell viability test

The purpose of this test is to determine whether the pair cell culture provides free radical resisting inductive Cytotoxic protection to extract of the present invention.THP-1 is a kind of human leukemia cell line (ATCC), wherein adds ferrous ion (as the oxidation stimulus object) and causes that by the Fenton response mechanism oxidative stress causes cell death (Figure 14).The interpolation of Black rice extract of the present invention reduces cell death in the mode that concentration relies on.

Embodiment 9: hepatic lipase suppresses

Extract of the present invention (being called as the 1st layer in Figure 15) suppresses the hepatic lipase activity.Hepatic lipase hydrolysis HDL phospholipid and triglyceride are inversely proportional to blood plasma HDL-C level and the post-heparin plasma hepatic lipase is active.Therefore, the result shows that the extract of the present invention that is rich in anthocyanin may increase HDL in the body.

Embodiment 10: prevent the generation of DNA otch

In this test, use hydrogen peroxide free radical and hydroxy radical, by using TAE buffer (40mM Tris-acetas, 2mM EDTA, 0.7% agarose gel electrophoresis that contains 0.5 μ g/ml ethidium bromide pH8.5) assess the effect of Black rice extract of the present invention in the prevention dna break.(UVP Inc., Upland CA) show the DNA band, with Lab Work software (UVP Inc., Upland, CA) analytic band density with desk-top ultraviolet diaphane.Be calculated as follows the inhibition that the DNA otch produces:

%Inh=(D _Natural-D _Sample)/(D _Natural-D _Oxidation) * 100

D _Natural, D _Oxidation, D _SampleRespectively the representative do not contain AAPH DNA, contain the DNA of AAPH and contain AAPH and the density of the DNA of testing sample.

The integrity of DNA is most important for cell division and survival.Shown that DNA is subject to oxidative damage in vivo, cause destroying transcribe, translation and dna replication dna, finally sudden change and cell death.For example, people such as Schneider [19] prove that hydroxyl radical free radical participates in DNA damage, points out early stage hydroxyl radical free radical to have biology related with mutation and oncogenic process.Transition metal-ascorbic acid-hydrogen peroxide system produces the sequence dependent DNA damage, the guanine preferential oxidation, and it is metal and the bonded result in DNA specific region that the part of prompting active oxygen composition produces.Former report shows, it is a kind of active useful means [20] that antioxidant antagonism active oxygen composition inducing DNA degenerates (comprising hydroxy radical and hydrogen peroxide free radical) people such as (, 1999) Kitts that can be used for estimating that the DNA otch produces test.

In this research, we have adopted the notion of non-locus specificity and locus specificity hydroxy radical, and use it in the dna break analysis (Figure 10 and 11).Obviously, no matter be that the site-specific right and wrong of going back are site-specific, hydroxyl radical free radical can both destroy the integrity (the 2nd road of Figure 10) of super coiled DNA chain.

Produce in the test at the inductive DNA otch of this hydroxyl radical free radical, the Black rice extract is higher than locus specificity free radical (table 3) to the prevention of non-locus specificity free radical.

The inhibition percentage ratio of table 3. Black rice extract to preventing that hydroxyl radical free radical inducing DNA otch from producing

????μg/ml	Anthocyanin content (%) 1	Non-site-specific	Site-specific
				Complete rice 100 1000 fractions 10 100 Trolox 0.17 1.7	???1.36±0.01 ???0.16±0.02	????65.7 ????83.9 ????54.4 ????82.8 ????44.0 ????80.8	????51.4 ????74.1 ????49.0 ????69.5 ????25.7 ????61.0

¹With anthocyanidin-3-glucoside and peonidin-outer standard of 3-glucoside conduct, measure by LC-MS.

It is also to be noted that, prevent super coiled DNA by hydroxyl radical free radical induce produce otch also with Black rice in anthocyanin distribute relevantly, the Black rice skin that promptly contains the high level anthocyanin is compared the ability higher (table 3) that suppresses the damage of hydroxyl radical free radical inducing DNA with complete rice.Standard anti-oxidant Trolox also shows similar trend, promptly to the inhibition higher (table 3) of non-locus specificity hydroxyl radical free radical institute inducing DNA damage, confirms that sequence signature is relevant with the inductive DNA chain interruption of hydroxyl radical free radical.The meaning that hydroxyl radical free radical inducing DNA otch produces is that may have Fenton reacted constituent such as hydrogen peroxide and transition metal ions in nucleus, it induces the cytotoxicity relevant with dna break.

Table 4. Black rice and anthocyanin are preventing that the super coiled DNA chain from suffering the effect aspect the fracture of hydrogen peroxide free yl induction

Sample	?％Inh
		The Black rice fraction, 1 μ g/ml, 10 μ g/ml, 25 μ g/ml, 100 μ g/ml	?1.0±0.7 ?7.4±0.3 ?25.8±8.7 ?82.2±7.8
Anthocyanidin-3-glucoside: peonidin-3-glucoside 9: 14: 11: 11: 49: 1	?63.2±1.6 ?50.4±2.1 ?38.7±0.3 ?50.2±0.6 ?40.7±0.0
		Trolox????10μg/ml	?100

In our research, we find that also the mode that the Black rice extract relies on concentration suppresses super coiled DNA fracture (table 4), still has significant effect when being low to moderate 10 μ g/ml.As mentioned above, anthocyanidin-3-glucoside and peonidin-3-glucoside exists as main pigment in Black rice.In our research, we find that anthocyanidin-3-glucoside and peonidin-3-glucoside show 11.5% and 11.4% the protection to the DNA chain interruption respectively when 1 μ g/ml, and protection brings up to 45.3% and 44.1% respectively when 5 μ g/ml levels.In the dna break model of this hydrogen peroxide free yl induction, also assessed the effect of anthocyanidin-3-glucoside and peonidin-3-glucoside combination.The result shows that along with the reduction of anthocyanidin-3-glucoside percentage ratio, protection also reduces (Figure 10 and table 3).The concentration dependent that uses the Black rice extract also to observe the super coiled DNA fracture suppresses (Figure 11 and table 4).

Embodiment 11: the anti-inflammatory test

A) the Black rice extract is in the effect that prevents aspect the nitric oxide

B) to the Western engram analysis of iNOS

A)

Mouse macrophage RAW 264.7 (ATCC) in the DMEM culture medium of having replenished 10% hyclone and antibiotic (100U/ml penicillin and 100U/ml streptomycin) at 37 ℃ and 5%CO ₂The middle cultivation.With cell with 2 * 10 ⁵The density of cells/well is inoculated in 96 orifice plates.After the incubated overnight, when cell attachment arrives the bottom, hole, add Black rice extract and 1 μ g/ml bacteria lipopolysaccharide (LPS, escherichia coli, serotype 0111:B4) that different content is dissolved in PBS, continued 24 hours.Culture medium five equilibrium (100 μ l) in other one 96 orifice plate, is added 100 μ l Greiss reagent (5% phosphoric acid and the 0.1% naphthyl ethylenediamine dihydrochloride aqueous solution that contain 1% sulfanilamide, 1: 1 v/v) [18] then.Read the absorbance that the plate device is measured 540nm with ELISA.Concentration according to the standard curve determination nitrite that obtains with same process.Calculate nitric oxide production inhibition according to following formula:

% inhibition=(Abs _Positive-Abs _Sample)/(Abs _Positive-Abs _Negative) * 100

Abs wherein _Positive, Abs _Negative, Abs _SampleRepresentative contains LPS, do not contain the culture medium of LPS and contains the absorbance of the sample of LPS respectively.

B)

In cell harvesting to 2 * sample reduction buffer, incubation is 5 minutes in boiling water.With 20 μ l sample pipetting volumes to 8%SDS-PAGE, electrotransfer (Bio-Rad Laboratory) to nitrocellulose filter subsequently.In containing the 50mM Tris buffer (pH7.5) of 150mM NaCl and 0.05%Tween-20, with 3% defatted milk powder closing membrane 1 hour at room temperature.Then film and mouse anti iNOS antibody (Pharmingen Transduction Laboratories) and mouse anti alpha-tubulin antibody (Sigma) in containing the 50mM Tris buffer (pH7.5) of 150mM NaCl 4 ℃ be incubated overnight.The goat anti-mouse IgG of film and horseradish peroxidase (Pharmingen Transduction Laboratories) incubation 1 hour at room temperature then.With 4-chloro-1-naphthols and hydrogen peroxide display target albumen [19] (people such as Bollag, 1996).

Except the active oxygen composition, be considered to energy induced mutation [21] (Keefer and Wink, 1996) as nitric oxide and metabolite peroxynitrite salt isoreactivity oxygen composition thereof.Two types nitric oxide synthetic (NOS) is arranged in mammalian cell, i.e. composing type NOS (cNOS) and induced NOS (iNOS), latter's most probable is by endotoxin such as lipopolysaccharide (LPS) and cytokine activation.INOS does not exist in most cells under normal operation, yet, after suitably stimulus object such as LPS and cytokine effect, can rapid induction and produce a large amount of nitric oxide.The incubation of bacteria lipopolysaccharide and macrophage is to estimate the inducible nitric oxide synthase to produce nitric oxide production a kind of fast method.People such as Leeuwenburgh [22] (1997) report contains significantly more 3-nitrotyrosine from the LDL of atherosclerosis of aorta inner membrance than blood plasma LDL, shows that the active nitrogen composition participates in aorta LDL oxidation and atherosclerosis.And verified, iNOS inhibitor N-imino group ethyl-L-lysine can limit existing atherosclerotic progress in the hypercholesterolemia rabbit, prevents atherosclerotic progress thereby point out inhibition iNOS to have to be beneficial to.

The mouse macrophage RAW264.7 of incubation can the great expression nitric oxide altogether with antibacterial LPS.Utilize Griess reagent, according to the metabolite nitrite indirect determination nitric oxide in the cell culture medium supernatant.Find that nitrite is 25 μ M in the culture medium, be higher than the macrophage of handling without LPS, show when stimulating the RAW264.7 cell, to have promoted nitric oxide production formation with bacteria lipopolysaccharide.

Add the generation that the Black rice extract has significantly reduced nitrite, show and suppressed NO production in the activated macrophage (Figure 18).It should be noted that also the inhibition that nitric oxide produces is not owing to any cytotoxicity, because cell viability normal (Figure 18) behind the interpolation Black rice extract.Also found nitric oxide production inhibition for anthocyanidin-3-glucoside and peonidin-3-glucoside.Under identical working condition, anthocyanidin-3-glucoside and peonidin-3-glucoside is displayed in Table 5 nitric oxide production inhibition in the activatory RAW264.7 cell of LPS.

The nitric oxide production inhibition percentage ratio that produces among the activatory mouse macrophage RAW264.7 of table 5. couple LPS (meansigma methods ± SD)

	Anthocyanidin-3-glucoside	Peonidin-3-glucoside
			????1μM	???12.7±2.7	???14.4±0.6
????10μM	???23.1±0.9	???22.3±3.7
			????100μM	???72.9±4.8	???35.0±2.5

It should be noted that when 100 μ M anthocyanidin-3-glucoside shows that the inhibition that nitric oxide is formed is higher than peonidin-3-glucoside.

In our research, obviously in the RAW264.7 cell, add LPS and can significantly induce the iNOS protein expression, this confirms (Figure 19) through the western blotting.The expression of adding Black rice extract demonstration iNOS protein (MW130kDa) in this cell culture model is suppressed (Figure 19) in the mode that concentration relies on.In addition, show with the cell lysate of anti-alpha-tubulin antibody trace, through or without LPS and sample treatment, this special protein all remains unchanged, therefore point out the Black rice specificity to suppress the expression of inducible nitric oxide synthase in macrophage, thereby reduce NO production under this condition.

The minimizing of the atherosis development of embodiment 12:Apo-E mice medium-sized artery

The ApoE deficient mice of C57BL/6J background is raised under routine raising condition available from Jackson Laboratories (BarHarbor, Maine U.S.A).The C57BL/6J mice is from Sun-Yat Sen medical university animal center.

Altogether 45 4 age in week male apoE deficient mice be divided into 3 groups at random, 15 every group, body weight coupling.Mice is accepted the purified diet (being summarized in the table 6) based on the AIN-93G goods, raises with one of following diet for every group: AIN-93G purified diet (positive group); The AIN-93G purified diet, it contains 5g/100g extract of the present invention: Black rice fraction (BRF group); The AIN-93G purified diet, it contains the white rice fraction of 5g/100g (WRF group).15 male 4 week C57BL/6J Mus in age (matched group) are in contrast raised the purified diet with AIN-93G.

With Oleum Glycines the protein in the different diet, fat and energy level are adjusted to same level (table 7) by adding casein.The composition of BRF and WRF is listed in table 5.All groups are all raised in the plastics cage with rustless steel graticule mesh top.Unrestrictedly give food and distilled water.16 weeks of Therapy lasted.In 4 groups, the average dietary intake ≈ 2.8g/d of every mice.Mice is weighed weekly twice in experimentation.

When experiment finished, all mices did not spend the night to food, got blood from the socket of the eye metaplexus under anesthesia, and it is killed benignly.Under 4 ℃,, be used for measuring the serum lipid level with 2800 * g low-speed centrifugal serum 20 minutes.

Table 6

The AIN-93 purified diet that is used for the laboratory rodent

Composition	G/kg food
		Corn starch	????397.486
Casein	????200
		The corn starch of dextrinize	????132
Sucrose	????100
		Oleum Glycines (not containing additive)	????70
Fiber	????50
		Mixture of inorganic substance (AIN-93G-MX) 1	????35
Vitamin mixtures (AIN-93-MX) 2	????10
		The L-cysteine	????3
Choline bitartrate (41.1% choline) 3	????2.5
		Tertiary butylated hydroquinone	????0.014

^1，2ICN?Company，U.S.A

³Molecular weight based on free alkali

Table 7

The main nutrient of different diet groups

Composition	Matched group	Positive group	The BRF group	The WRF group
					Energy (kcal/kg food)	????3766.0	????3766.0	??3766.0	??3766.0
Carbohydrate (g/kg food)	????629.486	????629.486	??629.486	??629.486
					Protein (g/kg food)	????200	????200	??200	??200
Total fat (g/kg food)	????70	????70	??70	??70

^*Matched group: raise C57BL/6J mice with AIN-93 G purified diet

Positive group: raise apoE deficient mice with the AIN-93G purified diet

BRF group: raise apoE deficient mice with the AIN-93G purified diet that contains 5g/100g Black rice fraction

WRF group: raise apoE deficient mice with the AIN-93G purified diet that contains the white rice fraction of 5g/100g

Table 8

The composition of Black rice and white rice fraction

Composition	Black rice fraction unit/100g	White rice fraction unit/100g
			Protein, g	????13.90	????12.20
Fat, g	????13.20	????14.10
			Carbohydrate, g	????47.36	????50.95
Moisture, g	????9.80	????7.96
			Fiber, g	????8.32	????7.04
Salt mixture, mg	????7420	????7750
			????P	????1694.10	????1542.50
????Ca	????60.20	????45.30
			????K	????673.70	????624.60
????Mg	????79.40	????80.40
			????Na	????2.11	????4.35
????Fe	????16.46	????6.30
			????Zn	????8.96	????4.92
????Mn	????11.67	????7.93
			????Mo	????0.33	????0.28
????Cu	????1.49	????0.91
			????Se	????0.15	????0.06
Vitamin, mg
			Vitamin B1	????2.30	????1.20
Vitamin B2	????0.40	????0.14
			Vitamin E	????0.60	????0.03
Nicotinic acid	????21.00	????13.00
			Total flavones, mg	????6.4	????1.2

Serum lipid distributes

Utilize Hitachi automatic analyzer (Tokyo) to measure serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C).Utilize cholesteryl esterase and cholesterol oxidase to test and measure serum TC.Measure the serum-concentration of HDL-C with Same Way.Calculate serum LDL concentration according to the Friedwald formula.

Atherosclerotic assessment

The quantitative of atherosclerosis fat bar carries out (18) by the pathological changes size of calculating in the aortic sinus as previously mentioned.In brief, from animal, take out heart and aortal epimere, the fat around careful the removing.Epimere is embedded in the O.C.T chemical compound, frozen in-20 ℃.Get aortic sinus (400um) section, take out every a slice (10um is thick) and analyze.The distal part of aortic sinus distinguishes that according to Tricuspid valve this is the junction of aorta and heart.The frozen section oil red O stain, and use haematoxylin redyeing.Utilize the direct photographic images of RGB camera that is connected with Olympus BX-50 optical microscope, and it is presented at Trinitron ^TMOn the RGB monitor, estimate the oil red O stain zone of each aortic valve section.Utilize Optima ^TM4.1 software (Image Processing Solutions) carries out graphical analysis.The result is expressed as by the percentage ratio of total cross section blood vessel wall area of oil red O stain (normal+disease district/section, as not comprise inner chamber).

Statistical analysis

The result is expressed as meansigma methods ± SD, determines difference by step ANOVA associating Student-Newman-keuls (SNK) multiple comparisons check.It is significant that the difference of P＜0.05 is considered to.

The result

Body weight

Experimental program begins with every group of 15 mices.The mice body weight in when beginning experiment is 17 ± 1g (meansigma methods ± SD).Last average weight is 25 to 27g.The significant difference (table 9) of not observing body weight in experimentation (Figure 16).

Table 9

Raise with the AIN-93G diet or contain the body weight of separate groups of mice in 16 weeks of the AIN-93G diet of WRF, BRF

Group	N	4 weeks	8 weeks	12 weeks	16 weeks
						Matched group	15	?17.07±1.08	?22.41±2.13	?25.67±2.34	?26.45±2.63
Positive group	15	?17.16±1.04	?21.13±3.14	?23.77±2.75	?24.47±2.85
						The BRF group	15	?17.11±1.05	?21.35±3.31	?24.73±3.24	?25.90±3.13
The WRF group	15	?17.18±1.07	?21.83±2.72	?24.38±3.07	?26.18±3.00

Serum cholesterol level

Serum TC in the matched group, LDL-C, HDL-C and LDL/HDL are different from other three groups (P＜0.05).TC, LDL-C in the BRF group and LDL/HDL level are lower than positive group and WRF group (P＜0.05), do not have difference between positive group and the WRF group.Although BRF and WRF group all have the HDL-C level that is higher than positive group, the BRF group contains the LDL/HDL (table 10) of reduced levels.

Table 10. is raised with the AIN-93G diet or is contained the serum lipid concentration of mice in 16 weeks of the AIN-93G diet of WRF, BRF ^*

Group	??n	??TC(mmol/I)	??LDL-C(mol/l)	??HDL-C(mmol/I)	?LDL/HDL
						Matched group	??10	??2.881±0.758a	??0.280±0.083a	??1.776±0.439a	?0.160±0.038a
Positive group	??10	??15.682±2.644b	??1.427±0.420b	??2.546±0.442b	?0.570±0.188b
						The BRF group	??10	??12.118±1.833c	??0.747±0.251c	??2.659±0.398c	?0.343±0.170c
The WRF group	??10	??17.240±3.646b	??1.315±0.562b	??2.645±0.663c	?0.450±0.179b

^*Numerical value is meansigma methods ± S.D.There is not the numerical value of common letter to have significant difference, P＜0.05 in the row.Used abbreviation: TC, T-CHOL; LDL, low-density lipoprotein cholesterol; HDL, HDL-C; C, cholesterol.

The development that the aortic sinus medium-sized artery is atherosis

After intervening for 16 weeks, in the matched group aortic sinus of raising, there is not visible atheromatous plaque with the AIN-93G diet.But visible atheromatous plaque in various degree in other aortic sinus of three groups.Speckle in positive group and WRF group than more serious (table 11) (Figure 17) in the BRF group.

The mean patch area of BRF group mice is lower than positive group and WRF group (P＜0.01).Raise with the plaque area of the aortic sinus of the mice of BRF and hang down 48.42% and 46.08% respectively than positive group and WRF group.The plaque area of positive group and WRF group mice does not have significant difference.

Table 11

After 16 weeks, raising with the AIN-93G diet or containing in the mice of AIN-93G diet of WRF, BRF the atheromatous plaque of aortic sinus ^*

Group	????N	Atherosclerosis area (%)
			Matched group	????15	??0a
Positive group	????15	??26.25±9.2b
			The BRF group	????15	??13.54±4.1c
The WRF group	????15	??25.11±7.15b

^*There is not the numerical value of common letter to have significant difference, p＜0.01 in the row.

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Claims (25)

1. one kind is extracted the method for compositions that contains anthocyanin from Black rice (Oryza sativa L), comprising:

A) skin of separation, hulling Black rice and starchy endosperm;

B) in separated outer layer, add at least a organic solvent and sour solution;

C) from separated outer layer, filter and remove and desolvate and acid, the chromogenesis fraction;

D) component of separation pigment fraction; With

E) therefrom collect the anthocyanin compositions.

2. the process of claim 1 wherein that physical property is separated skin and starchy endosperm in step a).

3. the process of claim 1 wherein and in step a), separate skin and starchy endosperm by the Black rice physical property of the shelling of milling.

4. the process of claim 1 wherein that organic solvent is selected from alcohol, ketone, hydrocarbon and water.

5. the process of claim 1 wherein that organic solvent is to have structure RCOR ¹Ketone, wherein R and R ¹Be C ₁-C ₆Alkyl.

6. the process of claim 1 wherein that organic solvent is C ₅-C ₁₀Hydrocarbon.

7. the process of claim 1 wherein that organic solvent is that to be selected from general formula be R-CHOHR, R-CH ₂The alcohol of OH and RCOH, wherein R is C ₁-C ₄Alkyl.

8. the process of claim 1 wherein that acid is selected from hydrochloric acid, acetic acid, citric acid, low-concentration sulfuric acid and tartaric acid.

9. the process of claim 1 wherein the acid that adds capacity, making pH is 1-4.

10. the process of claim 1 wherein that separated outer layer was dipped in the solvent 1-10 hour.

11. the process of claim 1 wherein and in step c), desolvate and acid by evaporating to remove.

12. the process of claim 1 wherein the component of in step d), separating pigment fraction by classification.

13. according to each the compositions of method preparation of claim 1-9.

14. a compositions, it contains anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside.

15. the compositions of claim 14, wherein the ratio of anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside is 10: 1.

16. the compositions of claim 14, wherein the ratio of anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside is 6: 1.

17. the compositions of claim 14, wherein the ratio of anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside is 3.5: 1 to 6.5: 1.

18. the compositions of claim 14, wherein the ratio of anthocyanidin-3-O-glucoside and peonidin-3-O-glucoside is 4: 1.

19. the compositions of claim 11 wherein also contains one or more antioxidants.

20. the compositions of claim 11 wherein also contains one or more sterols.

21. the compositions of claim 11 wherein also contains one or more stanols.

22. the compositions of claim 11, wherein also contain one or more antioxidants, it is selected from: vitamin E, beta-carotene, superoxide dismutase, catalase, glutathion peroxidase, glutathion reductase, tea catechin, chelating agen such as citric acid, EDTA, phenylalanine, phosphoric acid, tartaric acid and tryptophan; The chemical compound of preferential oxidation is as ascorbic acid, sodium sulfite and sodium sulfite; Water solublity chain terminating agent such as mercaptan and fat-soluble chain terminating agent such as alkyl gallates, ascorbic palmitate, tertiary butylated hydroquinone, butylated hydroxyanisol, Yoshinox BHT, hydroquinone, nordihydroguaiaretic acid and alpha-tocopherol.

23. a treatment or the prevention cardiovascular disease that comprises atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress of animal and the method for underlying diseases thereof comprise animal are used compositions according to the method preparation of claim 1.

24. treat or the cardiovascular disease that comprises atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress of prevention animal and the method for underlying diseases thereof, comprise the compositions of animal being used claim 11 for one kind.

25. treat or the cardiovascular disease that comprises atherosclerosis, inflammation, hyperlipemia, hypoalphalipoproteinemia, hypercholesterolemia and oxidative stress of prevention animal and the method for underlying diseases thereof, comprise the compositions of animal being used claim 12 for one kind.

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